Project Summary: Alzheimer?s disease is a widespread neurodegenerative disease which kills more people than prostate and breast cancer combined. There are currently no therapies to prevent or cure Alzheimer?s disease in spite of numerous attempts to develop treatments. The amyloid hypothesis states that Alzheimer?s disease begins when a small protein fragment known as amyloid beta forms toxic oligomers and aggregates. Amyloid beta fragments are created when the amyloid precursor protein transmembrane fragment, C99, is cleaved in the membrane by gamma secretase. Cholesterol has long been implicated as generally pro-AD and cholesterol has also been shown to increase the production of amyloid beta, but it is not known how cholesterol or cholesterol-rich lipid rafts increase C99 cleavage by gamma secretase. Several laboratories have shown cholesterol can form a complex with C99 and this complex is thought to increase amyloid beta production via co-localization of C99 and gamma secretase in lipid raft Nano domains. Lipids in rafts are thought to be more ordered than bulk (disordered phase) lipids and this order is here hypothesized to alter membrane protein structure. We want to study the impact of cholesterol and cholesterol-rich lipid rafts on C99 structure and proteolytic processing by gamma secretase. In Aim 1, we will determine the structure of C99 in an unpublished lipid raft-like bicelle (a detergent micelle with a bilayered core) via solution NMR. The C99 model generated from NMR will then be verified by EPR. In Aim 2, we will examine the impact of cholesterol and cholesterol-rich lipid rafts on gamma secretase processing of C99 with detailed kinetic studies. We will examine the impact of cholesterol on C99 processing in the context of a bulk lipid and as a C99 complex. The impact of cholesterol will be examined in liquid disordered and liquid ordered vesicles. Membrane thickness and lipid order are two defining features of lipid rafts and we will examine the impact of both parameters on gamma secretase- mediated C99 cleavage. Ideally, changes in C99 processing due to cholesterol and cholesterol-rich lipid rafts will be explained by the C99 structural model generated in Aim 1. Understanding of how and when cholesterol and lipid rafts impact C99 processing is important for understanding the success and failure of future Alzheimer?s disease therapies. In addition to studying an important problem related to Alzheimer?s disease, this proposal facilitate training for my goal of becoming a staff scientist. This proposal will allow me to become a well-rounded membrane protein structural biologist who is able to use enzymology and mammalian cell work to interpret structural meaning.